Various publications, which may include patents, published applications, technical articles and scholarly articles, are cited throughout the specification in parentheses, and full citations of each may be found at the end of the specification. Each of these cited publications is incorporated by reference herein, in its entirety.
Cytotoxic T lymphocytes (CTL) play an important role in the mammalian immune reaction to foreign materials and are capable of inducing the death of tumor cells in vivo. CTL are derived from naïve CD8+ T cells and recognize antigenic peptides presented by Major Histocompatibility Complex (MHC) class I cell surface receptors, also referred to as human leukocyte antigens (HLA). Naïve T cells are distinguished from activated T cells in that they have not yet encountered an antigen or other signal required for activation. It is generally accepted that two signals are required for induction of naïve T cells. Signal 1 is induced by the interaction between the T Cell Receptor (TCR) and the MHC/antigenic peptide complex and is aided by binding of CD8 co-receptors to non-polymorphic regions of MHC class I molecules. Signal 2 is qualitatively different from Signal 1 and is delivered via T cell co-stimulatory molecules interacting with complementary ligands on Antigen Presenting Cells (APC) that express MHC class II and co-stimulatory molecules. Signals 1 and 2 function synergistically and trigger a series of signalling events which ultimately induce T cells to proliferate, produce cytokines, and differentiate into antigen-specific CTL that can then travel throughout the body to search for and destroy other specific antigen-positive cells.
In addition it has been demonstrated that responses to cellular antigens are dependent on help delivered by CD4+ T cells, i.e., Helper T Lymphocytes (HTL). The nature of this help has been interpreted as the need for activated HTL to produce IL-2 necessary for CTL expansion. Recent studies have also shown that this help results from the activation of dendritic cells by HTL and is mediated via the interaction of CD40 and its ligand. Dendritic cells have been shown to be highly potent inducers of CTL responses and are thought to be the principal APC involved in priming CTL. It is generally accepted that APC, through mechanisms unique to these cells, take up antigens either in the form of soluble antigen associated with chaperone molecules or by phagocytosis.
In recent years, many genes encoding tumor associated antigens (TAA) that can be recognized by CTL have been identified from cDNA of a variety of human tumor cells. For example, the identification of TAA in melanoma has led to clinical trials to test therapies that target cancer cells using vaccination strategies in which the antigens are delivered in an immunogenic context in an attempt to induce potent T cell responses in vivo. These vaccination strategies with TAA hold promise for the development of novel cancer immunotherapies.
Adoptive immunotherapy is another strategy that holds promise as a novel cancer immunotherapy. Adoptive immunotherapy involves in vitro activation and expansion of T cells specific for one or more tumor antigens and subsequent treatment of patients with the activated T cells. Compared to vaccination therapies using TAA, adoptive T cell therapy has advantages because it involves the removal of T cells from the host environment where tolerogenic mechanisms can affect the immunogenic response. Furthermore, studies in mouse tumor models have demonstrated that adoptive immunotherapy can be efficacious with minimal toxicity. In the past, one obstacle in applying this strategy to the treatment of human tumors was the lack of information about immunogenic antigens that would render tumor cells susceptible to CTL-mediated destruction. More recently, however, isolation of tumor-reactive T cells from cancer patients has led to the identification of TAA to which CTL are directed. Some of these include tyrosinase, MART-1/Melan A, gp100, and MAGE. Of these, tyrosinase and MART-1 are nearly universally expressed on melanoma cells and therefore represent a desired target choice for adoptive immunotherapy for patients with melanoma.
Early adoptive immunotherapy approaches used Lymphokine-activated killer cells (LAK) and later tumor-infiltrating lymphocytes (TIL), both activated ex vivo with IL-2. The demonstration of efficacy was equivocal, however, and thus these early controlled clinical trials failed to show an advantage to the use of the ex vivo-activated cells over the direct administration of IL-2 to melanoma patients. More recent studies have clearly demonstrated the potential for certain adoptive T-cell therapeutic approaches (Yee et al., PNAS, Vol. 99, pp. 16168-16173, (2002); Dudley et al., Science, Vol. 298, pp. 850-854, (2002)). These studies involved use of either T-cell clones specific for MART-1 or gp100 and low-dose IL-2, or TILs expanded ex vivo with allogeneic feeder cells and high-dose IL-2. These studies confirmed that adoptive immunotherapy holds promise as a treatment for cancer.
The use of artificial antigen presenting cells (aAPCs) is an ex vivo method to reproducibly generate therapeutic numbers of antigen specific CD8+ T cells. For although it is possible to use naturally occurring APCs for T cell activation in vitro (e.g., dendritic cells, macrophages, B-cells, or autologous tumor cells), the efficiency of activation can be low since the MHC molecules of naturally occurring APCs contain many other peptide epitopes. As a result, there may be minimal presentation of selected epitopes. In addition, most of these presented peptides represent normal, innocuous endogenous proteins. A more direct approach to this problem is to activate CD8+ T cells specifically to only those epitopes relevant to combating the disease. This approach is accessible using aAPCs (See e.g. U.S. Pat. Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S. Patent Application Publication Nos. 2009/0017000 and 2009/0004142; and International Publication No. WO2007/103009).
One such aAPC has been developed utilizing a Drosophila melanogaster (fruit fly) embryonic cell line, which expresses the major histocompatibility complex (MHC) Class I molecules. Drosophila lacks homologues to human TAP1 and TAP2 peptide transporters, which are involved in the loading of peptide epitopes into the human MHC molecules. As a result, transfected Class I molecules and Class II molecules appear on the Drosophila cell surface as empty vessels. By incubating Drosophila cells transfected with MHC Class I- or MHC Class II-encoding expression vectors with one or more exogenous synthetic peptides that bind to the specific MHC molecules (i.e., TAA for presentation as T-cell peptide epitopes), all of the available MHC molecules may be occupied with MHC-restricted, specific peptide epitope(s). In particular, the high density expression of HLA-A2.1 MHC Class I molecules presenting single or multiple peptide epitopes, and the addition of key assisting molecules B7-1 (CD80), LFA-3 (CD58), ICAM-1 (CD54), and CD70 on these Drosophila aAPCs, permits the in vitro generation of potent, autologous cytotoxic CD8+ T cells which are specific for the selected peptides and suitable for use as a cell therapy.
One such cell therapy comprises an autologous immunotherapeutic product prepared with ex vivo-activated autologous CD8+ CTL exhibiting peptide specificity for selected HLA-A2.1-restricted peptides from melanoma-associated antigens. The active component of the cell therapy product is the patient's own CD8+ cells, which have been activated ex vivo by exposure to selected peptide-loaded aAPCs having specificity for the selected HLA-A2.1 restricted peptides. To generate the cell therapy product, the CTL are: 1) derived from autologous naïve T cells isolated from lymphapheresis samples harvested at a clinical site; 2) primed ex vivo against melanoma antigenic peptide epitopes using Drosophila cells as the aAPCs; 3) expanded by restimulation with autologous monocytes loaded with the melanoma antigenic epitopes preferably in the presence of both Interleukin-2 (IL-2) and Interleukin-7 (IL-7), followed by non-specific expansion using OKT®3; 4) harvested, washed, and re-suspended in final formulation for infusion; and, 5) infused into the patient from which the CD8+ cells were derived. The final cell therapy product for re-infusion preferably contains 1-10×109 CTL cells in 300 mL of Lactated Ringer's Injection Solution (76% v/v), 5% dextrose in normal saline (D5NS) (4% v/v), and human serum albumin (HSA) (20% v/v).
These promising new immunotherapies utilizing specific antigens for ex vivo-activation of autologous CD8+ CTL offer a promising strategy for the treatment of cancer. This is an especially exciting development for cancers that are incurable with current therapies. Multiple myeloma (MM) is a clonal B cell malignancy with an incidence of approximately 15,000 new cases per year in the United States. MM has a median survival of only three years and is characterized by proliferation and accumulation of mature plasma cells (PC) in the bone marrow (BM) leading to bone destruction, BM failure, anemia, and reduced immune function. MM remains essentially incurable by conventional anti-tumor therapy (Kyle and Rajkumar, N Engl J Med. 2004 Oct. 28; 351(18):1860-73. The identification of myeloma-specific antigenic peptides uniquely presented on multiple myeloma cells is an important step in the development of an effective immunotherapy treatment for MM.